The PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q96 ID for the applications described in the kit handbooks. If the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are used with other than QIAGEN kits, it is the user's responsibility to validate the performance of such product combination for any particular application.

Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori. |Analysis of multiple contiguous CpG sites. Methylation at nine independent CpG sites (highlighted in gray) is quantified in a single Pyrosequencing run. Position-specific information in the context of an analyzed sequence presents broad sequence methylation patterns. Note the built-in quality control sites (highlighted in yellow) consisting of cytosines converted to thymines, demonstrating full bisulfite conversion of the treated DNA.|Detection of tri- and tetra-allelic SNPs can be difficult with commonly used methods. This series of Pyrograms illustrates the ease of Pyrosequencing based detection of a tri-allelic SNP (red outline). C, T and G are serially dispensed in the Pyrosequencing reaction and only the incorporated nucleotides will elicit a signal peak. The result is a different peak pattern for homozygous samples of each allele (upper three Pyrograms) or compound peak patterns for heterozygous samples (lower three Pyrograms).||||||Linearity of methylation quantification by Pyrosequencing. PCR products from varying mixtures of unmethylated genomic DNA and methylated DNA (EpiTect Control DNAs) were analyzed by Pyrosequencing. A tight correlation between the known percentage of methylated DNA in the mixtures (squares) and the methylation percentage reported by Pyrosequencing (triangles) was observed (r2 = 0.9962). The graph represents the quantification of methylation at a single CpG site in the p16 gene. |The PyroMark Q96 ID manages all steps necessary to rapidly sequence and analyze up to 96 samples. Simply design the necessary assay and run files, load your samples and reagents, and walk away. The PyroMark Q96 ID dispenses reagents and nucleotides to each well with precision and detects emitted light signals with sensitive CCD sensors.||By processing 96 samples in a single run, the PyroMark Q96 ID combines analysis versatility with high throughput. The PyroMark Q96 MD explands this throughput with a robotic module that enables hands-free processing of ten 96-well plates. New assay design can be done with the PyroMark Q24, which offers the same analysis versatility on a smaller throughput scale.|PyroMark Q96 Software and PyroMark supplementary software are user-friendly interfaces granting access to assay design, run setup, and powerful data analyses of the obtained results. The software are driven by dropdown menus that ensure the correct selection of parameters and analysis modes for any assay, enabling new users to perform Pyrosequencing runs almost immediately.|The PyroMark Q96 Vacuum Workstation enables conversion of PCR products into the single-stranded DNA needed as template for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process can be carried out for up to 96 samples in parallel and takes only a few minutes.|The components of the PyroMark Q96 ID System are designed to make your research workflow straightforward and efficient. Each step is supported by software, kits, reagents, and sample preparation instrumentation that are optimized for Pyrosequencing.|

Integrating detection and quantification of genetic variation into one powerful system, Pyrosequencing with the PyroMark platform outperforms other sequence-based solutions in the analysis of targeted short DNA sequences. For analysis of methylation in epigenetic studies, Pyrosequencing generates highly reproducible quantification of methylation frequencies at individual or consecutive CpG sites (see figure "Analysis of multiple contiguous CpG sites"), and can detect and quantify even small changes in methylation levels (see figure "Linearity of methylation quantification").

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q96 ID is a fully integrated system that provides real-time sequence information and is highly suitable for detection of genetic variations, genetic quantification, and short DNA sequencing. The following products are used in combination with PyroMark Q96 ID instrument: PyroMark Q96 Vacuum Workstation, PyroMark CpG SW, PyroMark Assay Design SW, PyroMark IdentiFire SW, PyroMark Gold Q96 Reagents, and PyroMark Control Oligo. Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q96 Vacuum Workstation.

Steps of the Pyrosequencing reaction:

Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure "Principle of Pyrosequencing — step 1").

Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide (see figure "Principle of Pyrosequencing — step 2").

Step 3: ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated (see figure "Principle of Pyrosequencing — step 3").

Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure "Principle of Pyrosequencing — step 5").

Streamlined workflow — from sample to result

The versatile PyroMark Q96 ID seamlessly integrates into epigenetics and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. This highly reliable instrument enables sequence-based quantification and detection of SNPs, insertion and deletion mutations, CpG sites, as well as generation of sequence information. The streamlined workflow means that results can be achieved faster.

Fast and easy sample preparation

From PCR product to single-stranded template ready for sequencing — up to 96 samples can be prepared in parallel using the PyroMark Q96 Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling and the actual "hands-on time" is less than 5 minutes.

Procedure

Fast and easy sample preparation

From PCR product to single-stranded template ready for sequencing — up to 96 samples can be prepared in parallel using the PyroMark Q96 Vacuum Workstation in less than 15 minutes. The workstation ensures easy handling, and the actual "hands-on time" is less than 5 minutes.

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Gold reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. The PyroMark Q96 ID enables powerful and versatile analysis of genetic and epigenetic variation, whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, or polymorphisms in forensic samples of mitochondrial DNA. In addition, because Pyrosequencing integrates sequence detection and quantification, the high analysis resolution can lead to new discoveries.

Software

Easy-to-use PyroMark Q96 ID Software 2.5

PyroMark Q96 ID software version 2.5 enables comprehensive analysis of your results. The recently updated software now contains four analysis modes: AQ (allele quantification), SNP (analysis of SNPs and InDels), CpG (methylation analysis), and SQA (sequence identification). These four different types of analyses can be performed on the same plate, in the same run.

PyroMark Q96 ID Software 2.5 version 2.5.10.7 is the application software for setting up, performing and analyzing runs on PyroMark Q96 ID instruments. The main feature of this release is 64bit compatibility with Windows 7 operating systems.

This software can only be downloaded by users with a registered PyroMark Q96 ID instrument.